Evaluation of Pakistani wheat germplasm for leaf rust resistance at various locations

341 entries comprising of 250 genotypes/lines and 91 gene differentials were tested for leaf rust ( Puccinia triticina Erik) in different ecological zones of Punjab during 2016–17 and 2017–18. Each entry was planted in a single 1 m long row and Morocco was used as a spreader. Data on leaf rust severity was recorded once in 3 rd week of March during both study years at all locations by following Modified Cobb Scale while the data was recorded three times on 2 nd , 22 nd and 29 th March during 2018 at Faisalabad location to study rust development pattern. The disease severity ranged from 0-100S during 2016–17 and from 0-80S


Introduction
Wheat is one of the most important cereals in the world. Since green revolution, there is many fold increase in wheat production especially in the developing world [1]. Many factors are responsible for limiting wheat production like heat stress, drought stress, diseases and insect pests etc. Among different diseases like bunts, rusts and other foliar diseases rusts have induced serious yield losses [2].
Chemical control of the rust is being practiced in developed countries whereas in the developing world, it is not affordable for the farmers. Hence, they depend upon genetic resistance [3,4] and development of resistant cultivars is considered the most effective way to manage the disease [5,6]. Identification of genetic variability for rust resistance and its use to develop resistant cultivars through schematic breeding is pre-requisite to develop rust resistant cultivars. During the post green revolution period, cultivars with different genes (Lr10, Lr13, Lr26 etc) have continuously been released across the world and eliminated with the passage of time [4,7,8].
Leaf rust caused by Puccina triticina is a serious threat and results in major yield losses to wheat production in temperate zone areas like Pakistan where climate is generally arid, characterized by hot summers and cool or cold winters and wide variations between extremes of temperature at given locations [9]. Wheat breeders mostly introduced wheat breeding material mostly depending upon varieties containing genes Lr 1, Lr 13 and Lr 26 in combination with the minor genes [4]. These varieties help to boost up wheat production due to higher yield potential and adaptability to rusts especially for leaf rust resistance. However due to emergence of new leaf rust races in a short time period, the breeders periodically deployed new rust resistance genes all over the world [10]. The resistance of cultivars developed with monogenic or vertical résistance is not durable, therefore now, the breeders are mainly depending on new sources of resistance to leaf rust based on minor genes which slows down the rust development and significantly reduce the losses due to rusts [11]. Breeders are looking for the genotypes with slow rusting mechanism for use in the breeding program. In previous years, many accessions with this type of resistance have been selected [12][13][14] and were used for developing back crosses, top crosses for pyramiding minor genes present in wheat germplasm [15] and many wheat varieties like Seher-06, Shafaq-06, Lasani-08, Faisalabad-08, AARI-11, Pb-11, Galaxy-13 etc have been released which contributed significantly in enhancing wheat production in the Punjab province and were cultivated in other provinces of Pakistan [4,16,17]. Breeders are now preferring genotypes with slow rusting or horizontal resistance based on minor genes or combination of minor and major genes [8,18]. Area Under Disease Progressive Curve (AUDPC) has been used by many scientists to understand disease development pattern and they preferred the genotypes having slow rusting pattern with lower AUDPC values for disease [19]. The current studies were designed to evaluate wheat germplasm for identification of sources of leaf rust resistance, to identify the resistance mechanism and pattern of rust development.in different ecological zones of Punjab-Pakistan.

i-Experimental material & locations
(29.3544˚N and 71.6911˚E) and Agriculture Adaptive Research Farm, Kot Naina district Narowal (32.1836˚N and 75.2523˚E) which were located in different agro-ecological zones of the Punjab having difference in temperature and rainfall pattern (Table 1).

ii-Experiment management
Each entry was sown in a 2 m long single row by maintaining 30 cm row to row distance. A single line of susceptible cultivar, Morocco was repeatedly sown after every 10lines of experimental material. Two rows of spreader "Morocco" were sown on each side of the experimental material to capture pathotype inoculum. The fertilizer NPK was applied as a basil dose at the rate of 120, 75 and 60 kg ha -1 . Three irrigations were applied at different growth stages i.e. first at tillering stage, second at booting stage and third at grain formation stage. Weeds were controlled by manual hoeing at all four locations.

iii-Inoculation of experimental material
The inoculation of material was done three times during first fortnight of February at an interval of five days at all locations. The previous years collected inoculums mixture (mixture of pathotypes found from all over Punjab) stored at -80˚C was used after proper heat shock and re-hydration process. 1 g of inoculum was mixed in 250 g of talcum powder and was dusted on experimental material.

iv-Data recording and analysis
At Faisalabad during the year 2017-2018, the leaf rust data was recorded three times at twoweek interval starting from 01-03-2018 to 29-03-2018. The data was used to calculate Area Under Disease Progressive Curve (AUDPC) [20][21][22]. The AUDPC was calculated by using Excel based software developed by CIMMYT [20].
Where Y i is disease severity at t i time, t (i+1) − t i is time interval between two consecutive data recording events, n is no. of data recordings.
AUDPC/Day was calculated by dividing AUDPC with the number of days counted from the first to the last data recording date. At all other three locations except, disease appearance was based on natural inoculation. The data was recorded once during 4 th week of March. The data was recorded following Modified Cobb's Scale and the entries were classified as resistant, moderately resistant, moderately susceptible, moderately resistant to moderately susceptible and susceptible [23].

Results
Screening of wheat germplasm for leaf rust resistance is a constant feature of our research program. In the current study, a set of 341 genotypes comprising of 250 varieties/advanced lines and 91 gene differentials were evaluated against leaf rust in field conditions. Tables 2-6 shows reaction of this set of genotypes to leaf rust at four locations viz. Faisalabad, Bahawalpur, Khanewal and Kot Naina during the study years.

i-1 st year results
During the year 2016-2017, there was no disease reaction on 17 entries (Table 3) at Faisalabad. Out of these, 2 were the advanced lines and remaining 15 were gene differentials. Similarly, there was no disease on 30 entries containing 2 advanced lines and 2 approved varieties. At Khanewal, there was no disease on 4 advanced lines, approved varieties and 28 gene differentials. At Kot Naina, disease did not appear on 58 entries and out of these, 19 were advanced lines/varieties and 39 were gene differentials. The differences in the response of different genotypes at different locations might be due to variation in environmental conditions as shown in Table 1 and variation in the natural inoculum prevailing at these locations. Two advanced lines V-15235 and HYT 60-5 and fifteen gene differentials did not show any disease symptoms whereas one gene differential Lr-29 was moderately susceptible on all four locations of Punjab, Pakistan during the year 2016-17 (Table 4). 6 gene differentials (Lr-10, Lr-11, Lr-14B, Lr-24, Lr-30 and Lr-34) showed susceptibility.

ii-2 nd year results
During the year 2017-2018, there was no disease appeared on 2 genotypes HYT 60-5 and Ujala-16 whereas 28 differentials were free from leaf rust at Faisalabad (Table 5). At Bahawalpur, 58 entries did not show any disease reaction and out of these 12 were advanced lines/varieties and rests were the gene differentials. At Khanewal, 69 entries were disease free. Among these 69, 17 were advanced lines/varieties and 52 were gene differentials. At Kot Naina, 75 entries were completely free from leaf rust disease. Out of these, 18 were advanced lines/varieties and remaining were gene differentials.

iii-Future sources of resistance
The advance line HYT 60-5 had not shown disease symptom at any experimental location during both the study years (Table 7) in Punjab, Pakistan. Therefore, this can be treated as the most promising leaf rust resistant genotype for use in the breeding program. Similarly, the leaf rust gene differentials containing Lr-19, Lr-26 and Lr-27+31 genes did not show any susceptibility at any target location during both study years. Therefore, the material containing these 3 genes may be targeted for improving leaf rust resistance in bread wheat breeding material.

iv-Disease progression
Area under disease progression curve (AUDPC) show disease development in relation to time period and AUDPC/Day gives its clear picture. In the present study during the year 2017-18, AUDPC was calculated to find out disease progression pattern. AUDPC ranged from 0-550 while that of Morocco which is a susceptible check has AUDPC value of 600. 120 entries have disease progression 0 which shows there may be a major gene-based resistance in these entries. AUDPC/Day was calculated for the rest of 130 entries to have a deep understanding of the disease progression and out of which 43 entries have AUDPC/Day value ranging from 1-2 and 28 entries have AUDPC/Day value ranging from 2-3. These entries have slow rust development pattern than average which shows that these entries are extremely useful for use in breeding for durable rust resistance and can be utilized as a parent in back cross and top cross in breeding scheme.

Discussion
In Past, wheat varieties with different types of rust resistance mechanism have been released all over the world including Pakistan [24,25]. The varieties having monogenic and varietal resistance generally have short life whereas the varieties with multiple gene resistance and horizontal resistance have long life [1,11,14,15]. Furthermore, use of monogenic resistance also sometimes create monocentric situation. Material possessing 1B-1R translocation was used all over the world and results in the evolution of new devastating genes like Yr 9, Yr 27 and Ug99 [4,6,9]. Current studies revealed that most of the gene differentials studied showed virulence however virulence for Lr 19, Lr 26 and Lr 27+31 was not present in the Punjab, Pakistan. Therefore, the varieties having adult plant resistance based on minor genes were preferred by   showed moderately resistant reaction which shows that these lines have horizontal resistance based on minor genes. V-14154 has been approved as Akbar-19 due to its better performance in yield trials and disease screening nurseries at national level [25]. Presence of minor genes-based resistance has been reported in international and Pakistani germplasm by [15,26]. During 2017-18, there was no disease on HYT 60-5. Among approved wheat varieties, Ujala-16 has not shown any disease during 2017-18 whereas at Bahawalpur, Khanewal and Kot Naina, it showed moderately resistant moderately susceptible (MRMS) reaction. Variable disease reactions of different wheat genotypes have already been reported in literature due to different genetic background of breeding material and variation in environmental conditions [8,16].
As far as reaction of gene differentials is concerned, leaf rust resistant genes Lr19, Lr26, Lr 27+31 have not shown any virulence. Therefore, these genes can be used as a source of leaf rust resistance in wheat breeding program preferably in combination with other major and minor genes because it has been revealed that the cultivars having major and minor gene combinations have high and sustainable level of resistance which showed that variation in the disease progression level is due to the difference in host pathogen interaction [18].   (Table 8). AUDPC/Day value is the indicator of progression rate. The genotypes with lower AUDPC value have lesser disease development rate as compared to the genotypes with higher AUDPC/Day value. It was concluded that the genotypes having AUDPC/Day value less than 10 can be utilized as valuable source in the breeding program and such material with lower AUPDC values can be released for commercial cultivation as use of such material is environment friendly, enhances farmers profitability.

Conclusion
The experimental material showing wider range of diversity was evaluated in different agroecological zones having variation in climatic factors. The material was inoculated by the mixture of inoculum collected from different parts of Punjab and Sindh province during the previous three years. The gene differentials containing leaf rust genes Lr-19, Lr-26 and Lr-31 showed resistance against the leaf rust in different agro-ecological zones of the Punjab. Hence, the breeders can focus on the introgression of these genes into future breeding material. Among the tested advanced lines, V-14154, V-14124 and HYT 60-5 were very useful advanced lines for release as a commercial variety or use as a parent for leaf rust resistance as they showed good level of resistance against leaf rust. V-14154 (Akbar-19) has been released as a commercial cultivar on the basis of its higher yield, adaptability and disease resistance. Among  the current commercial varieties of the Punjab, Ujala-16 has shown the best resistance for leaf rust during study years.